Mouse anti camkii

Cells were rinsed with PBS and immediately fixed using 4% paraformaldehyde with 4% sucrose in PBS for 15 min. After rinsing with PBS, a 15-min permeabilization using 0.2% Triton X-100 in PBS, and a 1-h blocking solution (4% BSA and 4% normal goat serum in PBS), cells were incubated overnight at 4°C in primary antibody diluted in blocking solution (mouse anti-CaMKII from Millipore 05-532, rabbit anti-Gluc from Nanolight 401P, mouse anti-GAD67 from Millipore MAB5406, mouse anti-GFAP from Cell Signaling Tech. 3670, chicken anti-MAP2 from Abcam ab5392). After washing with PBS, the cells were incubated with a fluorescent secondary antibody and Hoeschst (Hoechest 33342 from Invitrogen, donkey anti-rabbit Alexa Fluor 488 from Invitrogen A21206, all others from Jackson ImmunoResearch). Coverslips were mounted onto glass slides using Prolong gold mounting solution (Invitrogen P36930). Imaging was performed using either a Nikon E800 epi-fluorescence microscope or Nikon A1 for confocal imaging.

Primary antibodies were used as follows: rabbit anti-Kirrel3 1:2000 (this study, generated against C-terminal peptide), rabbit anti-GABA 1:5000 (Sigma, St. Louis, MO, United States), goat anti-GFP 1:5000 (Abcam, Cambridge, MA, United States), guinea pig anti-VGLUT1 1:10,000 (Millipore, Billerica, MA, United States), mouse anti-MAGUK 1:1000 (NeuroMab, UC Davis/NIH NeuroMAB Facility, Davis, CA, United States), mouse anti-FLAG M2 1:5000 (Sigma), rabbit anti-synapsin 1:1000 (Millipore), rabbit anti-cFos 1:500 (Santa Cruz Biotech, Dallas, TX), rabbit anti-GFP 1:1000 (Invitrogen, Waltham, MA, United States), chick anti-FLAG 1:1000 (Gallus Immunotech, Cary, NC, United States), mouse anti-PSD95 1:2000 (Thermo Scientific), mouse anti-GAPDH 1:5000 (Millipore), chick anti-MAP2 1:10,000 (Abcam), rabbit anti-calretinin 1:2000 (Swant, Switzerland), mouse anti-parvalbumin 1:5000 (Swant), mouse anti-CamKII 1:5000 (Millipore), rat anti-Somatostatin 1:500 (Chemicon), rabbit anti-calbindin d28k 1:2000 (Swant), rabbit anti-VIP 1:500 (Immunostar, Hudson, WI, United States). All secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, United States) and used at 1:1000.